Development and use of a multiplex PCR to detect common mastitis pathogens in ewe’s milk

1. Development and use of a multiplex PCR to detect common mastitis pathogens in ewe’s milk Emma Monaghan

2. Aim of Project • To develop and assess the use of a multiplex PCR as a tool to identify bacterial species commonly associated with mastitis in sheep.

3. Objectives of Project • To develop an effective technique for the isolation of DNA directly from sheep milk for use in a multiplex PCR. • To develop a multiplex PCR for the most commonly recognised mastitis pathogens: • Staphylococcus aureus • coagulase-negative staphylococci • Escherichia coli • Mannheimia haemolytica • Streptococcus agalactiae • Streptococcus dysgalactiae • Streptococcus uberis using specific primers to amplify products of varying fragment sizes.

4. Objectives of project continued • To use the multiplex PCR technique developed to process approximately 500 sheep milk samples and analyse the occurrence of the previously specified mastitis-associated bacterial species. • To look at the infection status of sheep over time.

5. Mastitis Background • Defined as ‘inflammation of the udder’, usually as a result of infection with pathogenic bacteria. • Defined by severity and clinical signs into clinical, subclinical and chronic infection.

6. Impact of Mastitis Direct effects: • Temporary of permanent reduction in milk production • Milk quality reduction • Animal welfare issue/ quality of life: Mastitis can be very painful. Affected ewes become uninterested in their lambs • Premature culling and death as a result of infection Economic: • Annual loss of £300 million to UK dairy farmers • Prevention costs: antibiotics, disinfectants • Treatment costs: drugs, veterinary costs etc

7. Mastitis pathogens in sheep • Staphylococcus aureus • coagulase-negative staphylococci • Escherichia coli • Mannheimia haemolytica • streptococcal species- • Streptococcus agalactiae, • Streptococcus dysgalactiae • Streptococcus uberis.

8. Multiplex PCR • Multiplex PCR works by combining multiple primer sets specific for individual sequence targets in a single PCR mixture. • This produces amplicons of variable sizes, allowing the identification of targeted bacterial pathogens in a milk sample.

9. Primers • Specific primers aiming to combine in a multiplex PCR are targeting: E.coli, Mannheimia haemolytica, Staphylococcus aureus, coagulase-negative staphylococci, Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis. • So far, I have optimised and tested the specificity of a mixture of previously published and validated primers and newly designed ones.

10. Specificity results designed primers 1

11. Specificity results designed primers 2

12. Specificity results of designed primers 3 KEY  primer amplified DNA of bacterial species specific amplification of bacterial species X no amplification seen TBC to be confirmed

13. Summary of progress • DNA isolation directly from milk technique has been identified, tested and optimised. • Successfully tested the DNA isolation technique with universal and specific primers sets. • Have Streptococcus uberis and Streptococcus dysgalactiae specific primer sets. • Continue to design, optimise and test primer sets, in addition to testing of previously published and validated primers to combine in the multiplex PCR.

14. Thanks for listening! Any questions?